This invention relates to the treatment of grafts prior to transplantation into a host. More specifically, this invention is directed to incubating or perfusing a graft such as tissue or an organ with TGF-.beta. and transplanting the treated graft into a suitable recipient.
The success of a transplant of an allograft in a host depends on such factors as the antigens on the transplanted tissue that are recognized by the recipient as foreign and can evoke the rejection response, the cells in the recipient's immune system that mediate rejection, and the reactions that modify either the presentation of the foreign antigen or the cellular response.
The major histocompatibility complex (MHC) is the most important immunogenetic system encoding transplantation antigens. The MHC is genetically complex because it includes many different loci, each encoding separate cell-surface antigens, and because the loci have extensive polymorphism. The loci of the MHC fall into one of two classes, Class I or Class II, based on their tissue distribution, the structure of the expressed antigens, and their functions. Class I antigens, present on all nucleated cells, serve as the primary targets for cytotoxic T (CD8.sup.+) lymphocytes. Class II antigens are not distributed in the tissue as widely and serve as primary targets for helper T (CD4.sup.+) lymphocytes.
The polymorphic forms of the individual loci of human leukocyte antigen (HLA), the MHC in humans, have been recognized by antibodies and by various in vitro techniques that measure T-lymphocyte recognition. These responses, mediated by the recipient's recognition of polymorphism in the donor, correlate with the strong rejection reactions that take place in vivo.
Investigation into the cellular basis of graft rejection, using both in vitro and in vivo studies, reveals that both CD4.sup.+ and CD8.sup.+ lymphocytes participate in the rejection response.
Attempts to prolong the survival of allografts and xenografts after transplantation, both in experimental models and in medical practice, have centered mainly on the suppression of the immune apparatus of the recipient. This treatment has as its aim preventive immunosuppression and/or treatment of graft rejection.
Examples of agents used for immunosuppression include cytotoxic drugs, antimetabolites, corticosteroids, and antilymphocytic serum. Nonspecific immunosuppressive agents found particularly effective in preventive immunosuppression (azathioprine, bromocryptine, methylprednisolone, prednisone, and most recently, cyclosporin A) have significantly improved the clinical success of transplantation. The nephrotoxicity of cyclosporin A after renal transplantation has been reduced by coadministration of steroids such as prednisolone, or prednisolone in conjunction with azathioprine. In addition, kidneys have been grafted successfully using anti-lymphocyte globulin followed by cyclosporin A. Another protocol being evaluated is total lymphoid irradiation of the recipient prior to transplantation followed by minimal immunosuppression after transplantation.
Treatment of rejection has involved use of steroids, 2-amino-6-aryl-5-substituted pyrimidines, heterologous anti-lymphocyte globulin, and monoclonal antibodies to various leukocyte populations, including OKT-3. See generally J. Pediatrics, 111: 1004-1007 (1987), and specifically U.S. Pat. No. 4,665,077.
The principal complication of immunosuppressive drugs is infections. Additionally, systemic immunosuppression is accompanied by undesirable toxic effects (e.g., nephrotoxicity when cyclosporin A is used after renal transplantation) and reduction in the level of the hemopoietic stem cells. Immunosuppressive drugs may also lead to obesity, poor wound healing, steroid hyperglycemia, steroid psychosis, leukopenia, gastrointestinal bleeding, lymphoma, and hypertension.
In view of these complications, transplantation immunologists have sought methods for suppressing immune responsiveness in an antigen-specific manner (so that only the response to the donor alloantigen would be lost). Such specific immunosuppression generally has been achieved by modifying either the antigenicity of the tissue to be grafted or the specific cells capable of mediating rejection. In certain instances, whether immunity or tolerance will be induced depends on the manner in which the antigen is presented to the immune system. Pretreating the allograft tissues by growth in tissue culture before transplantation has been found in two murine model systems to lead to permanent acceptance across MHC barriers. Lafferty et al., Transplantation, 22: 138-149 (1976); Bowen et al., Lancet, 2:585-586 (1979). It has been hypothesized that such treatment results in the depletion of passenger lymphoid cells and thus the absence of a stimulator cell population necessary for tissue immunogenicity. Lafferty et al., Annu. Rev. Immunol., 1: 143 (1983). See also Lafferty et al., Science, 188: 259-261 (1975) (thyroid held in organ culture) and Gores et al., J. Immunol., 137: 1482-1485 (1986) and Faustman et al., Proc. Natl. Acad. Sci. U.S.A., 78: 5156-5159 (1981) (islet cells treated with murine anti-Ia antisera and complement before transplantation). Also, thyroids taken from donor animals pretreated with lymphocytotoxic drugs and gamma radiation and cultured for ten days in vitro were not rejected by any normal allogeneic recipient. Gose and Bach, J. Exp. Med., 149: 1254-1259 (1979). All of these techniques involve depletion or removal of donor lymphocyte cells.
In some models such as vascular and kidney grafts, there exists a correlation between Class II matching and prolonged allograft survival, a correlation not present in skin grafts. Pescovitz et al., J. Exp. Med., 160:1495-1508 (1984); Conti et al., Transplant. Proc., 19:652-654 (1987). Therefore, donor-recipient HLA matching has been utilized. Additionally, blood transfusions prior to transplantation have been found to be effective. Opelz et al., Transplant. Proc., 4: 253 (1973); Persijn et al., Transplant. Proc., 23: 396 (1979). The combination of blood transfusion before transplantation, donor-recipient HLA matching, and immunosuppression therapy (cyclosporin A) after transplantation was found to improve significantly the rate of graft survival, and the effects were found to be additive. Opelz et al., Transplant. Proc., 17: 2179 (1985).
The transplantation response may also be modified by antibodies directed at immune receptors for MHC antigens. Bluestone et al., Immunol. Rev. 90:5-27 (1986). Further, graft survival can be prolonged in the presence of antigraft antibodies, which lead to a host reaction that in turn produces specific immunosuppression. Lancaster et al., Nature, 315:336-337 (1985).
The immune response of the host to MHC antigens may be modified specifically by using bone marrow transplantation as a preparative procedure for organ grafting. Thus, anti-T-cell monoclonal antibodies are used to deplete mature T cells from the donor marrow inoculum to allow bone marrow transplantation without incurring graft-versus-host disease. Mueller-Ruchholtz et al., Transplant Proc., 8:537-541 (1976). In addition, elements of the host's lymphoid cells that remain for bone marrow transplantation solve the problem of immunoincompetence occurring when fully allogeneic transplants are used.
The survival time of skin grafts has been prolonged by a factor of two by treatment in vitro with cortisone, thalidomide, or urethane before implantation into a laboratory animal. The amount of drug locally applied to the skin was smaller than the amount required to achieve a similar effect by injecting the drug systemically. In an additional study, the donor skin was treated in vitro with streptokinase/streptodornase, or with RNA and DNA preparations of the recipient. Further, treatment of transplant tissues with a solution of glutaraldehyde prior to transplantation was found to reduce their antigenicity. See U.S. Pat. No. 4,120,649.
The transforming growth factor-.beta. (TGF-.beta.) molecules identified thus far are each dimers containing two identical 112 residue polypeptide chains linked by disulfide bonds. The molecular mass of these dimers is about 25 kd. Biologically active TGF-.beta. has been defined as a molecule capable of inducing anchorage-independent growth of target cell lines or rat fibroblasts in in vitro cell culture, when added together with EGF or TGF-.alpha. as a co-factor. TGF-.beta. is secreted by virtually all cell types in an inactive form. This latent form is first activated by proteolytic cleavage of mature TGF-.beta. from its precursor (at the Arg-Ala bond in position 278). A non-covalent complex is formed from the association of the mature TGF-.beta. with the precursor remainder. This complex is disrupted so as to activate the TGF-.beta. either by exposure to transient acidification or by the action of exogenous proteases.
There are at least three forms of TGF-.beta. currently identified, TGF-.beta..sub.1, TGF-.beta..sub.2, and TGF-.beta..sub.3. Suitable methods are known for purifying this family of TGF-.beta.s from platelets or placenta, for producing it in recombinant cell culture, and for determining its activity. See, for example, R. Derynck et al., Nature, 316:701 (1985) and U.S. Ser. Nos. 715,142; 500,832; 500,833, all abandoned; European Pat. Pub. Nos. 200,341 published Dec. 10, 1986, 169,016 published Jan. 22, 1986; 268,561 published May 25, 1988; and 267,463 published May 18, 1988; U.S. Pat. No. 4,774,322; Seyedin et al, J. Biol. Chem., 262: 1946-1949 (1987); Cheifetz et al, Cell, 48: 409-415 (1987); Jakowlew et al., Molecular Endocrin., 2: 747-755 (1988); and Dijke et al., Proc. Natl. Acad. Sci. U.S.A., 85: 4715-4719 (1988), the entire contents of these publications being expressly incorporated by reference.
TGF-.beta. has been shown to have numerous regulatory actions on a wide variety of both normal and neoplastic cells. Recent studies indicate an important role for TGF-.beta. in cells of the immune system (J. Kehrl et al., J. Exp. Med., 163:1037 [1986]; H-J. Ristow, Proc. Natl. Acad. Sci. U.S.A., 83:5531 [1986]; A. Rook et. al., J. Immunol., 136:3916 [1986]) and in proliferation of connective and soft tissue for wound healing applications (M. Sporn et al., Science, 219:1329 [1983]; R. Ignotz et al., J. Biol. Chem., 261:4337 [1986]; J. Varga et al., B. B. Res. Comm., 138:974 [1986]; A. Roberts et al., Proc. Natl. Acad. Sci. U.S.A., 78:5339 [1981]; A. Roberts et al., Fed. Proc., 42:2621 [1983]; A. Roberts et al., Proc. Natl. Acad. Sci. U.S.A., 83:4167 [1986]; U.S. Ser. No. 500,833, supra; U.S. Pat. No. 4,774,228 to Seyedin et al.), as well as epithelia (T. Matsui et al., Proc. Natl. Acad. Sci. U.S.A., 83:2438 [1986]; G. Shipley et al. Cancer Res., 46:2086 [1986]). Moreover, TGF-.beta. has been described as a suppressor of cytokine (e.g., IFN-.gamma., TNF-.alpha.) production, indicating its use as an immunosuppressant for treating inflammatory disorders (Espevik et al., J. Exp. Med., 166: 571-576 [1987]; European Pat. Pub. Nos. 269,408 published Jun. 1, 1988 and 213,776 published Mar. 11, 1987), and as a promoter of cachexia (Beutler and Cerami, New Eng. J. Med., 316: 379 [1987]). Further, TGF-.beta. induces collagenase secretion in human fibroblast cultures (Chua et al., J. Biol. Chem., 260:5213-5216 [1983]); stimulates the release of prostaglandins and mobilization of calcium (A. Tashjian et al., Proc. Natl. Acad. Sci. U.S.A., 82:4535 [1985]); and inhibits endothelial regeneration (R. Heimark et al., Science, 233:1078 [1986]).
TGF-.beta. is multifunctional, as it can either stimulate or inhibit cell proliferation, differentiation, and other critical processes in cell function (M. Sporn, Science, 233:532 [1986]).
The multifunctional activity of TGF-.beta. is modulated by the influence of other growth factors present together with the TGF-.beta.. TGF-.beta. can function as either an inhibitor or an enhancer of anchorage-independent growth, depending on the particular set of growth factors, e.g., EGF or TGF-.alpha., operant in the cell together with TGF-.beta. (Roberts et al., Proc. Natl. Acad. Sci. U.S.A., 82:119 [1985]). TGF-.beta. also can act in concert with EGF to cause proliferation and piling up of normal (but not rheumatoid) synovial cells (Brinkerhoff et al., Arthritis and Rheumatism, 26:1370 [1983]).
Most recently, TGF-.beta. has been found to suppress the expression of Class II histocompatibility antigens on human cells induced by human interferon-.gamma. and to inhibit constitutive expression of the Class II antigen message in the cells (Czarniecki et al., J. Immunol., 140: 4217-4223 [1988]; Czarniecki et al. J. Interferon Res., 7: 699 [1987]; Palladino et al., J. Cell. Biochem., Supp. 11A [Jan. 17-Feb. 5, 1987], UCLA Symposia on Molecular and Cellular Biology, Alan R. Liss, Inc., New York, abstract A016, p. 10; Chiu et al., Triennial Symposium: Biology of Growth Factors, University of Toronto, Ontario, Canada, [Jun. 17-19, 1987]; Palladino et al., Immunobiology, 175: 42 [1987]).
For a general review of TGF-.beta. and its actions, see Sporn et al., J. Cell Biol., 105: 1039-1045 (1987) and Sporn and Roberts, Nature, 322: 217-219 (1988).
There is a need in the art for a method to prolong graft survival in transplant operations and minimize the toxicity and other adverse effects arising from the use of large doses of immunosuppressants.
Accordingly, an object of this invention is to provide for longer graft survival in the host.
Another object is to provide for a transplantation method wherein lower amounts of immunosuppressive agents, if any, need be administered to the host to achieve successful results, thereby reducing the side effects associated with systemic administration of immunosuppressive drugs.
These and other objects will become apparent to one of ordinary skill in the art.